Modified U7 Snrna Construct

Loss of nuclear TDP-43 is observed in a number of diseases or disorders including >95% of all Amyotrophic Lateral Sclerosis (ALS) and tau-negative Frontotemporal Dementia (FTD) cases. This results in the inclusion of cryptic exons (CE) with subsequent functional loss of important disease-modifying genes, due to the absence of TDP-43 repression of these cryptic exons. TDP-43 regulated cryptic exons in both STMN2 and UNC13A have been mechanistically linked to ALS and FTD: STMN2 and UNC13A encode an axonal and synaptic protein, respectively and are crucial for normal neuronal function. In both cases, loss of nuclear TDP-43 results in the incorporation of a CE during splicing resulting in the depletion of the full-length mRNA and reduction of functional protein expression. Loss of nuclear TDP-43 also results in aberrant RNA processing, with STMN2 being the most significantly affected. Its depletion results in impaired axonal regeneration, which is alleviated when STMN2 levels are restored. For UNC13A human genetic evidence supports its impact in disease aetiology: Intronic SNPs in UNC13A are the second strongest risk factor for sporadic ALS, are associated with reduced patient survival, and shown to directly enhance cryptic exon inclusion.

TDP-43 regulated cryptic exons (CEs) are also known to affect numerous other transcripts which have crucial neuronal functions. One such example is in the ELAVL3 gene which encodes for a neuronal-specific RNA binding protein. The ELAVL3 CE leads to protein loss, which has been documented in ALS post mortem neurons, and leads to alterations in neurite maturation, maintenance. Similarly, TDP-43 loss induces a CE and consequent loss of another neuronal-specific RNA binding protein, CELF5, loss of which is known to cause motor neuron degeneration in model systems. CEs also appears in the INSR transcript leading to its reduction, with insulin signalling having emerged as an important pathway for neuronal health and maintenance.

There is therefore a need to further understand the role of TDP-43 depletion in disease, and to generate new therapeutic approaches for alleviating diseases associated with TDP-pathology, including but not limited to neurodegeneration, particularly in ALS/FTD.

According to a first aspect of the present invention, is provided a modified U7 snRNA construct comprising

The antisense sequence directs the construct to the TDP-43 regulated cryptic exon sequence or flanking regions thereof, while the sequence comprising a binding domain for hnRNP is capable of recruiting a hnRNP protein, and more particularly an endogenous hnRNP protein in a cell, to pre-mRNA containing the cryptic exon. Importantly, binding of the hnRNP protein acts to repress splicing of the cryptic exon, even in the absence of TDP-43 binding, or in cells depleted of TDP-43, such that the cryptic exon is at least partially excluded in the mature RNA of the cell transcript. This restores the functionality of genes containing TDP-43 regulated cryptic exons, e.g., in cells depleted of TDP-43. The constructs herein can therefore be used to further probe, understand, or treat diseases or disorders characterised by TDP-43 dysfunction or pathology.

According to a second aspect of the present invention, is provided a vector that comprises or encodes for the modified U7 snRNA construct of the first aspect. In some embodiments, the vector is a viral vector.

According to a third aspect of the present invention, is provided a pharmaceutical composition comprising one or more of the constructs according to the first aspect, and/or one or more of the vectors according to the second aspect.

According to a fourth aspect of the present invention, is provided the construct of the first aspect, the vector of the second aspect or the pharmaceutical composition of the third aspect for use in therapy. Also disclosed herein is the construct of the first aspect, the vector of the second aspect or the pharmaceutical composition of the third aspect for use as a medicament, for use in the manufacture of a medicament, or for use in a method of treatment (e.g., of a neurodegenerative or muscular disease or disorder).

According to a fifth aspect of the present invention, is provided the construct of the first aspect, the vector of the second aspect, or the pharmaceutical composition of the third aspect, for use in the treatment of a disease characterised by TDP-43 dysfunction. In some embodiments, the disease is a neurodegenerative or muscular disease. In some embodiments, the disease is selected from Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), Inclusion body myositis or myopathy (IBM), Alzheimer's disease, FOSMNN (Facial onset sensory and motor neuronopathy), Perry Syndrome, Limbic-Predominant Age-Related TDP-43 Encephalopathy (LATE) or a combination thereof.

According to a sixth aspect of the present invention, is a method of modulating splicing of a TDP-43 regulated cryptic exon, the method comprising delivering to a cell the construct of the first aspect, the vector of the second aspect, or the pharmaceutical composition of the third aspect, wherein the method comprises contacting the construct with a cell to modulate splicing of the TDP-43 regulated cryptic exon in the cell.

According to a sixth aspect of the invention, there is provided a combined vector comprising two or more of the constructs described herein or of the first aspect of the invention (i.e., in tandem, or one downstream of another, such that the combined vector comprises at least two constructs, each comprising one antisense sequence as defined herein and each comprising a sequence comprising a binding domains for a hnRNP protein as defined herein). In preferred embodiments, the two or more modified U7 snRNA constructs comprise different antisense sequences that are capable of binding to (i.e., they are at least 90%, or at least 95%, or 100% complementary to) different TDP-43 regulated cryptic exons described herein. In some embodiments, the combined vector may comprise three or more constructs as defined herein. In some embodiments, the combined construct comprises two or more antisense sequences that are complementary (i.e., at least 90% complementary, or at least 95% complementary, or 100% complementary) to two or more TDP-43 regulated cryptic exon sequences or flanking regions thereof. In some embodiments, the TDP-43 regulated cryptic exon is selected from one of the TDP-43 regulated cryptic exons defined herein. In some embodiments, each antisense sequence is a sequence that is complementary (i.e., 90%, 95% or 100% complementary) to SEQ ID NO: 1, 2, 3,4, 7, 9, or 448-453). In some embodiments, at least one of the antisense sequences, or each antisense sequences, is complementary to a TDP-43 binding region of the TDP-43 regulated cryptic exon, preferably wherein at least one of the antisense sequences, or each antisense sequence, is complementary (i.e., 90%, 95% or 100% complementary) to SEQ ID NO: 12, 23-26 or 32. In some embodiments, the combined vector comprises a construct as defined herein comprising an antisense sequence which is at least 90% complementary to a UNC13A TDP-43 regulated cryptic exon or flanking region thereof and a construct as defined herein comprising an antisense sequence which is at least 90% complementary to a STMN2 TDP-43 regulated cryptic exon or flanking region thereof. In some embodiments, the combined vector comprises a construct as defined herein comprising an antisense sequence which is at least 90% complementary to a UNC13A TDP-43 regulated cryptic exon or flanking region thereof and a construct as defined herein comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a INSR TDP-43 regulated cryptic exon or flanking region thereof. In some embodiments, the combined vector comprises a construct as defined herein comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a STMN2 TDP-43 regulated cryptic exon or flanking region thereof and a construct as defined herein comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a INSR TDP-43 regulated cryptic exon or flanking region thereof. In some embodiments, the combined vector comprises a construct comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a UNC13A TDP-43 regulated cryptic exon or flanking region thereof, a construct comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a STMN2 TDP-43 regulated cryptic exon or flanking region thereof, and a construct comprising an antisense sequence which is at least 90% complementary (or 95%, or 100% complementary) to a INSR TDP-43 regulated cryptic exon or flanking region thereof.

In some embodiments, the combined vector comprises two or more constructs defined herein, wherein the two or more sequences comprising a binding domain for a hnRNP protein may be according to any sequence as described herein. In some embodiments, the two or more sequences comprising a binding domain for a hnRNP protein may be different or identical. In some embodiments, the two or more sequences comprising a binding domain for a hnRNP protein may be a binding domain for a hnRNP A or hnRNP H protein, and in some examples, a hnRNP A protein.

In some embodiments, the combined vector comprises two or more promoter sequences, wherein the two or more promoter sequences are upstream of each construct. The promoters may be any promoter sequence used in the art. In some embodiments, each of the two or more promoter sequences are the same or different. In some embodiments, the combined vector comprises two or more 3′ box sequences, wherein the two or more 3′ box sequences are downstream of each construct. The 3′ box sequences may be the same or different and may be any 3′ box sequence used in the art.

In some embodiments, the combined vector comprises two or more U7 cassettes, wherein each cassette comprises a promoter, a modified U7 snRNA construct as defined herein, and a 3′ box sequence, wherein the promoter is upstream of the modified U7 snRNA construct and the 3′ box sequence is downstream of the modified U7 snRNA construct. In some embodiments, the combined vector comprises a stuffer sequence between each of the two or more U7 cassettes. The stuffer sequences serve to space out the two promoters. The stuffer sequence may be any suitable stuffer sequence used in the art.

In some embodiments, the combined vector comprises (from upstream to downstream) at least a:

The present inventors have developed tools that can target TDP-43 regulated cryptic exons and modulate their aberrant cryptic splicing in cells (e.g., upon depletion of TDP-43). The modulation of splicing means that splicing of the cryptic exon is at least partially repressed which in turn means that inclusion of the TDP-43 regulated cryptic exon in mature RNA is at least partially prevented, leading to the formation of a correctly spliced mature RNA transcript which can be translated into a fully functional protein. This therefore restores the production of functional proteins encoded by genes that contain TDP-43 regulated cryptic exons.

There are a number of TDP-43 regulated cryptic exons that are aberrantly spliced upon depletion of TDP-43 in the nucleus. TDP-43 depletion is associated with a number of diseases including neurodegenerative and muscular diseases, including ALS and FTD as described in the background section of this application. TDP-43 regulated cryptic exons are characterised by a TDP-43 binding region either within the cryptic exon or in close proximity to the cryptic exon (i.e., in the flanking regions of the cryptic exon), said TDP-43 binding region typically being UG rich. During normal splicing (i.e., in healthy cells), TDP-43, which is a transcriptional repressor protein, binds to the TDP-43 binding domain and represses splicing of the cryptic exon; this has the effect that the cryptic exon is not included in the mature mRNA of the transcript and a functional protein is produced. However, depletion of TDP-43 from the nucleus of cells means that the cryptic exon sequence is aberrantly spliced; this has the effect that the cryptic exon is included in the mature mRNA of the transcript meaning functional protein is not produced.

The constructs, vectors and pharmaceutical compositions disclosed herein can crucially be used to at least partially, or in some instances substantially completely or completely, restore correct splicing in the absence of TDP-43. The U7 constructs disclosed herein comprise both (i) an antisense sequence that guides the U7 snRNP to bind to the target cryptic exon (i.e., present in the pre-mRNA) and (ii) an hnRNP binding sequence for recruitment of an endogenous hnRNP protein. The tethering of hnRNPs substitutes for the loss of TDP-43 allowing for at least partial abolishment the cryptic splicing event. This restores the “normal” protein production which occurs in healthy cells (i.e., without TDP-43 depletion of dysfunction). This approach is particularly effective because hnRNPs are ubiquitously expressed and hence the constructs can be used in all cells that express them. It is particularly surprising that the tethering and recruitment of a hnRNP protein can almost completely substitute for the loss of TDP-43 function in repression of cryptic exons. Such an effect is found to be more pronounced and most effective with the highly endogenously expressed proteins such as hnRNP A1, with hnRNP H also showing good efficacy.

To the present inventor's knowledge, there has been no modified U7 snRNA constructs targeting TDP-43 regulated cryptic exons in the prior art, nor would it have been expected that recruiting a hnRNP A1 protein to a TDP-43 regulated cryptic exon (i.e., in the pre-mRNA) with a U7 construct would be sufficient to rescue the loss of TDP-43 binding in a TDP-43 depleted cell, given TDP-43's widespread binding in and/or across the suppressed cryptic exon. While other modified U7 constructs have been previously used in gene therapy, such constructs have a different target and a different mode of action. Instead, modified U7 snRNA constructs of the prior art seek to target standard constitutive exons or constitutive exons that are alternatively spliced due to mutations in the DNA, rather than cryptic exons, let alone constructs used to rescue splicing of TDP-43 regulated cryptic exons. The difference is that TDP-43 regulated cryptic exons are non-conserved intronic sequences that are erroneously included in mature RNA in cells depleted of TDP-43. These differ from typical constitutive exons which are instead supposed to be included in mature RNA. Previous U7 modified constructs therefore had a different aim, to promote exon inclusion and reduce gene expression of various genes. This is different to the constructs of the present invention which instead repress splicing of the cryptic exon to restore expression of TDP-43 regulated genes. The prior art constructs also have completely different targets and therefore completely different uses. No prior art constructs have been used to correct TDP-43 regulated cryptic exons, to rescue the correct splicing of genes which are depleted in the cell (e.g., due to TDP-43 pathology).

The construct in accordance with the present invention may be referred to as a “bifunctional construct”. This “bifunctional” approach provides a modified U7 snRNA construct which comprises both (i) an antisense sequence which binds to the TDP-43 regulated cryptic exon or flanking regions thereof, and (ii) a binding sequence for an hnRNP protein to recruit an endogenous hnRNP. This is demonstrated to be more effective than analogous U7 snRNA constructs which only comprise an antisense sequence (i.e., in the absence of a hnRNP binding sequence, which may be referred to as “single” target constructs herein). The design and approach of the present invention also allows for more flexibility as the antisense sequence need not be restricted to targeting core splice elements (e.g., splice sites) for reinstalling splicing repression. Indeed, example constructs described herein are found to effectively correct splicing, despite comprising antisense sequences that target different regions of TDP-43 regulated cryptic exons. In some examples, the antisense sequence binds to a TDP-43 binding region of a TDP-43 regulated cryptic exon, while correcting splicing. Since TDP-43 has as repressive role in healthy cells, and blocks splicing machinery from recognising the cryptic exon, constructs comprising antisense sequences that target the TDP-43 binding region serve to provide a steric block within this region, which contributes to blocking cryptic splicing. In alternative examples, the antisense sequence binds to a splice site of the TDP-43 regulated cryptic exon while correcting splicing. Constructs comprising antisense sequences that target the splice sites means that the splice sites are masked and less available for splicing by the splicing machinery within the cell. Further, it is also demonstrated that correct splicing is restored when the antisense sequence binds to an exonic splice enhancer (i.e., as identified by ESE finder 3.0) located within the TDP-43 regulated cryptic exon. Since ESEs are motifs within the cryptic exon sequence that promote or enhance splicing, blocking these motifs blocks cryptic splicing of the cryptic exon sequence. The present inventors demonstrate that the constructs can target a wide range of different target sequences within the TDP-43 regulated cryptic exon and flanking regions thereof, while still being effective at correcting splicing. Further, the present inventors demonstrate that this approach can be used to effectively correct splicing, at least partially, of various TDP-43 regulated cryptic exons.

There is also no prior example of a U7 construct that aims to target and correctly splice a TDP-43 regulated cryptic exon, which comprises both an antisense sequence which targets the TDP-43 regulated exon and a binding domain for a hnRNP protein. Crucially, different to prior approaches, the binding domain for the hnRNP protein seeks to recruit a hnRNP protein which takes over the repressive function (e.g., in cells depleted of TDP-43) that TDP-43 normally has in “healthy cells”. While previous U7 constructs have been described that couple antisense sequences with a binding sequence for a protein, these have been used against a different gene target. Additional U7 constructs of the prior art have a different aim, that is, to promote inclusion of a constitutive exon in the resultant mRNA (e.g., due to a mutation in a gene which alters splicing), rather than repress the inclusion of a cryptic exon in the resultant mRNA, let alone a TDP-43 regulated cryptic exon. Finally, in some instances, other U7 constructs in the art have instead aimed to recruit exonic splicing enhancers, such as SR proteins. SR proteins have the opposite effect to recruitment of a hnRNP protein as described in the present invention, since hnRNP proteins instead have a repressive effect.

A major advantage of a using a modified U7 snRNA approach is that snRNPs naturally reside in the nucleus where cryptic exon splicing happens. This results in localisation of the antisense containing U7 snRNA in the cellular compartment where splicing needs to be corrected. The use of antisense sequences in snRNPs also provides enhanced stability of the resultant RNA-protein complexes with the pre-mRNA (i.e., which contains the cryptic exon).

Another advantage is that modified U7 snRNAs can be packaged into vectors, such as viral vectors, which enable long lasting manufacture of the gene therapy following a single injection. This allows cells to produce their own therapeutic molecules as a single dose gene therapy, and is therefore improved as compared to ASO approaches. These constructs also provide a more stable therapeutic approach as compared to ASO targeting which are more sensitive to degradation. The small delivery of the U7 expression gene also allows their delivery in combination with other antisense or supplemental gene constructs in a single viral vector or ITR cassette. Finally, it is hypothesised that the larger size of the modified U7 snRNA construct as compared to an ASO approach could, in some instances, be more effective at correcting splicing due to steric effects; this is since the constructs may also provide a more effective steric block which contributes to the repression of the cryptic splicing event.

Since aspects of the invention are demonstrated to at least partially correct the splicing of TDP-43 regulated cryptic exons, aspects of the present invention can therefore be used to probe TDP-43 pathology and/or the role of TDP-43 pathology in disease. For example, as TDP-43 clearance is happening in >95% of ALS cases this approach is applicable and beneficial for the vast majority of ALS patients.

The present inventors have also uniquely demonstrated that a vector comprising two or more of the constructs of the invention (i.e., in tandem, or one after each other) suppresses TDP-43 cryptic exon inclusion in different genes. Different from any prior approach, this combined construct is able to target and rescue splicing for multiple TDP-43 regulated cryptic exons in different genes. The combined construct showed similar suppression of three TDP-43 regulated exons, UNC13A, INSR and STMN2, as compared to individual construct transfection. The result is unexpected considering the combined construct comprises multiple (and in some examples, identical promoters) and surprising in the context of promoter competition and promoter interference given three identical promoters were used to drive the expression of three different antisense sequences.

In some embodiments, constructs of the invention can be used to correct splicing of the TDP-43 regulated UNC13A cryptic exon. This cryptic exon is found to cause UNC13A downregulation at the transcript and protein level and is detected specifically in patient post-mortem brain regions affected by TDP-43 proteinopathy or dysfunction, including both ALS and FTD. Further, this cryptic exon is also found to overlap with the disease-associated variant rs12973192 previously identified in multiple genome-wide association studies linked to ALS/FTD risk, as well as disease aggressiveness. The UNC13A cryptic exon is therefore associated with TDP pathology, and disease aggressiveness. Correcting splicing of the UNC13A gene can therefore be used to further understand and/or treat diseases associated with ALS and FTD, and SNPs (e.g., rs12973192) in the UNC13A gene.

In some embodiments, constructs of the invention can be used to correct splicing of the TDP-43 regulated STMN2 cryptic exon 2a. This is important considering loss of nuclear TDP-43 results in the incorporation of this cryptic exon during splicing resulting in the depletion of the full-length mRNA and reduction of functional protein expression. This effect is most pronounced for STMN2, where aberrant RNA processing results in impaired axonal regeneration. Correcting splicing of the STMN2 gene can therefore be used to further understand and/or treat diseases associated with TDP-43.

Embodiments of the present invention are also used to correct splicing of the TDP-43 regulated INSR cryptic exon (between INSR exons 6 and 7). The INSR CE leads to loss of the protein, which normally acts as a receptor for insulin. Insulin signalling plays an important role in neuronal maintenance, and restoration of INSR levels would contribute to an amelioration of neuronal homeostasis.

Embodiments of the present invention are also used to correct splicing of other TDP-43 regulated cryptic exons, such as the ELAVL3 CE, the G3BP1 CE, the AARS1 CE, the CELF5 CE, the CAMK2B CE or the UNC13B CE. Preventing cryptic splicing and restoration of these proteins is considered to be therapeutically beneficial. In particular, the ELAVL3 CE leads to alterations in neurite maturation and is implicated in ALS, while the CELF5 CE leads to motor neuron degeneration in model systems.

Also described herein is a modified U7 snRNA construct comprising

In some embodiments, the antisense sequence is at least 90% complementary to SEQ ID NO: 3 or 4.

Also described herein is a modified U7 snRNA construct comprising (i) an antisense sequence having between 16 to 30 nucleotides which are at least 90% complementary to a TDP-43 regulated cryptic exon sequence in STMN2 and flanking regions thereof, preferably wherein the antisense sequence is at least 90% complementary to SEQ ID NO: 7, and

Also described herein is a modified U7 snRNA construct comprising

Also described herein is a modified U7 snRNA construct comprising

Also described herein is a modified U7 snRNA construct comprising

Also described herein is a modified U7 snRNA construct comprising a modified Sm motif comprising

Also disclosed herein is a modified U7 snRNA construct comprising

Also disclosed herein is a system comprising a construct, vector, or pharmaceutical composition and a cell, wherein said cell comprises or expresses a hnRNP protein. The cell may be as elsewhere defined herein.

For any sequence disclosed herein, the complementary sequence and reverse complement sequence is also disclosed. Also disclosed herein is a vector or construct with a complementary sequence to that described herein which may be used to encode for the constructs described herein.

The terms “treatment” and “treating” herein refer to an approach for obtaining beneficial or desired results in a subject, which includes a prophylactic benefit and a therapeutic benefit.

“Therapeutic benefit” refers to eradication, amelioration or slowing the progression of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the patient may still be afflicted with the underlying disorder.

“Prophylactic benefit” refers to delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. In the context of the present invention, the prophylactic benefit or effect may involve the prevention of the condition or disease. The construct, vector, or pharmaceutical composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

The term “effective amount” or “therapeutically effective amount” refers to the amount of the construct, vector, or pharmaceutical composition needed to bring about an acceptable outcome of the therapy as determined by reducing the likelihood of disease as measurable by clinical, biochemical or other indicators that are familiar to those trained in the art. The therapeutically effective amount may vary depending upon the condition, the severity of the condition, the subject, e.g., the weight and age of the subject and the mode of administration and the like, which can readily be determined by one of ordinary skill in the art.

The term “subject” refers to any suitable subject, including any animal, such as a mammal. In preferred embodiments described herein, the subject is a human.

The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) includes those embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, that “consist of” or “consist essentially of” the described features. The term “comprises” or “comprising” can be used interchangeably with “includes”.

“Capable of binding” as described herein refers to any nucleotide sequence that binds to the stated target region (e.g., the pre-mRNA containing the TDP-43 regulated cryptic exon). This can be defined as any nucleotide sequence may be substantially complementary (e.g., at least 90% complementary, or at least 95%) or complementary (e.g., 100% complementary) to the target sequence and/or at least part of a splicing element which has the same number of nucleotides as the antisense sequence.

“Sequence identity” as described herein refers to the % degree of similarity between two nucleotide sequences of the same length.

“UNC13A” as defined herein is a gene that encodes for the UNC13A protein. UNC13 proteins play an important role in neurotransmitter release at synapses.

“STMN2” as defined herein is a gene that encodes for stathmin 2 protein. This protein plays a regulatory role in neuronal growth.

“INSR” as defined herein is a gene that encodes for an insulin receptor which is a member of the receptor tyrosine kinase family of proteins, where binding of insulin or other ligands to this receptor activates the insulin signalling pathway.